Immunglobulin M-degrading enzyme of Streptococcus suis (IdeSsuis): modulation of B-cell function and consequences for mucosal colonization

猪链球菌是猪最重要的病原体之一，也是一种新兴的人类病原体。在猪身上，猪链球菌非常有效地定植上呼吸道粘膜，包括鼻咽、扁桃体(假定的进入口)和咽后淋巴结。猪链霉菌是唯一一种表达免疫球蛋白(Ig)M降解酶的细菌，命名为IdeSsuis，我们鉴定为半胱氨酸蛋白酶。在前面的项目中，我们证明了IdeSsuis介导的IgM裂解是一种新的补体逃避机制，导致调理减少和提高细菌存活率。然而，我们发现IdeSsuis对IgM的切割在导致脑膜炎的侵袭性感染中并不是至关重要的。然而，IdeSsuis对IgM的切割可能与猪链球菌在黏膜表面的定植有关，这些粘膜表面的IgM浓度低于血清中的浓度。因此，我们假设IdeSsuis通过定植链球菌介导的IgM蛋白分解足以削弱该隔室中依赖IgM的效应器功能。用野生型和突变型猪链球菌对无猪链球菌的仔猪进行定植实验，以定量测定定植效率和粘膜表面的IgM降解。我们将验证这样的假设，即猪链球菌表达的IdeSsuis对表达膜IgM(即B细胞受体，BCR)的B细胞有效，特别是在定植的粘膜部位。最近，我们发现IdeSsuis不仅能裂解可溶性的IgM，而且还能裂解表达在常规B细胞(即B2细胞)和非常规类B1细胞上的IgM BCR。IdeSsuis对IgM和IgM-bcr的切割预计会导致黏膜B细胞反应缺陷，并减少对猪链球菌的抗体产生，尤其是在猪缺乏IGD的情况下。更详细地说，IdeSsuis可能会破坏猪链球菌与IgM-BCR的结合。随后，我们预计B细胞对猪链球菌的内化减少，这将导致MHC-II抗原呈递减少，并减少与淋巴结中滤泡辅助性T细胞(TFH)的相互作用。用猪链球菌微囊化或其非微囊化等基因突变体对无猪链球菌的仔猪上呼吸道进行实验定植，以分析被壳多糖激活的类B1细胞。在进一步的动物实验中，我们专门研究了猪链球菌定植通过IdeSsuis在黏膜部位切割IgM来调节获得性免疫的假说，从而导致攻击时保护性免疫受损。这项实验将包括对表达野生型IdeSsuis和缺乏IgM切割的IdeSsuis的点突变变体的最先进的互补猪链霉菌突变体进行比较。这一建议中产生的数据有望有助于更好地理解IgM降解在定植过程中的作用，并解释为什么在所有研究的猪链球菌血清型中，IgM蛋白降解是高度保守的表型。

Streptococcus suis is one of the most important porcine pathogens and an emerging human pathogen. In pigs, S. suis colonizes very efficiently the upper respiratory mucosa including nasopharynx, tonsils (the putative entry port), and retropharyngeal lymph nodes. S. suis is unique among bacterial pathogens in expressing an immunoglobulin (Ig) M degrading enzyme, designated IdeSsuis which we identified as a cysteine protease. In the preceding Project we demonstrated that IdeSsuis–mediated IgM cleavage is a novel complement evasion mechanism leading to reduced opsonisation and enhanced bacterial survival. We found, however, that IgM cleavage by IdeSsuis is not crucial during invasive infection leading to meningitis. However, cleavage of IgM by IdeSsuis might be relevant for S. suis colonization of mucosal surfaces where IgM concentrations are lower than in serum. Thus, we hypothesize that IdeSsuis–mediated IgM proteolysis by colonizing streptococci is sufficient to diminish IgM-dependent effector functions in this compartment. Colonization of S. suis-free piglets with wild-type and mutant S. suis strains will be induced experimentally to quantitatively determine colonization efficacy and IgM degradation on mucosal surfaces. We will test the hypothesis that S. suis-expressed IdeSsuis is effective on B cells expressing membrane IgM (i.e. B cell receptor, BCR) especially at mucosal sites of colonization. Recently we have generated evidence that IdeSsuis not only cleaves soluble IgM but also the IgM BCR expressed on conventional B cells (i.e. B2 cells) as well as on non-conventional B1-like cells. IgM and IgM-BCR cleavage by IdeSsuis is expected to result in a defective mucosal B cell response and reduced antibody production to S. suis, even more so as pigs lack IgD. In detail, IdeSsuis may disrupt the binding of S. suis to the IgM-BCR. Subsequently we expect reduced internalization of S. suis by B cells which should result in reduced antigen presentation on MHC-II and reduced interaction with follicular T helper (TfH) cells in the lymph node. Experimental colonization of the upper respiratory tract of S. suis-free piglets with encapsulated S. suis or its unencapsulated isogenic mutant will be conducted to analyze B1-like cells activated by capsular polysaccharides. In a further animal experiment we specifically investigate the hypothesis that S. suis colonization modulates adaptive immunity through IgM cleavage by IdeSsuis at mucosal sites leading to impaired protective immunity upon challenge. This experiment will include comparison of state-of-the-art complemented S.°suis mutants expressing either wild-type IdeSsuis or a point-mutated variant of IdeSsuis deficient in IgM cleavage. The data generated in this proposal is expected to lead to a better understanding of the role of IgM degradation during colonization and to explain why IgM proteolysis is a highly conserved phenotype in all investigated S. suis serotypes.